title: Multicenter evaluation of a standardized protocol for noninvasive gene expression profiling.
abstract: Gene expression profiling of transplant recipient blood and urine can potentially be used to monitor graft function, but the multitude of protocols in use make sharing data and comparing results from different laboratories difficult. The goal of this study was to evaluate the performance of current methods of RNA isolation, reverse transcription and quantitative polymerase chain reaction (qPCR) and to test whether multiple centers using a standardized protocol can obtain the same results. Samples, reagents and detailed instructions were distributed to six participating sites that performed RNA isolation, reverse transcription and qPCR for 18S, PRF, GZB, IL8, CXCL9 and CXCL10 as instructed. All data were analyzed at a single site. All sites demonstrated proficiency in RNA isolation and qPCR analysis. Gene expression measurements for all targets and samples had correlations >0.938. The coefficient of variation of fold-changes between pairs of samples was less than 40%. All sites were able to accurately quantify a control sample of known concentration within a factor of 1.5. Collectively, we have formulated and validated detailed methods for measuring gene expression in blood and urine that can yield consistent results in multiple laboratories.
authors.affiliation: Department of Immunology, Cleveland Clinic, Cleveland, OH, USA. keslark@ccf.org
authors.forename: K S
authors.initials: KS
authors.lastname: Keslar
authors.forename: M
authors.initials: M
authors.lastname: Lin
authors.forename: A A
authors.initials: AA
authors.lastname: Zmijewska
authors.forename: T K
authors.initials: TK
authors.lastname: Sigdel
authors.forename: T Q
authors.initials: TQ
authors.lastname: Tran
authors.forename: L
authors.initials: L
authors.lastname: Ma
authors.forename: M
authors.initials: M
authors.lastname: Bhasin
authors.forename: P
authors.initials: P
authors.lastname: Rao
authors.forename: R
authors.initials: R
authors.lastname: Ding
authors.forename: D N
authors.initials: DN
authors.lastname: Iklé
authors.forename: R B
authors.initials: RB
authors.lastname: Mannon
authors.forename: M M
authors.initials: MM
authors.lastname: Sarwal
authors.forename: T B
authors.initials: TB
authors.lastname: Strom
authors.forename: E F
authors.initials: EF
authors.lastname: Reed
authors.forename: P S
authors.initials: PS
authors.lastname: Heeger
authors.forename: M
authors.initials: M
authors.lastname: Suthanthiran
authors.forename: R L
authors.initials: RL
authors.lastname: Fairchild
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D005786:Gene Expression Regulation; D006801:Humans; D016030:Kidney Transplantation; D057230:Limit of Detection; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014184:Transplantation, Homologous
chemical_list: D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase